1. Field of the Invention
Embodiments of the present invention relate to the field of biotechnology. More particularly, embodiments of the present invention relate to proteins and cells expressing such proteins which are useful in prolonging cell viability at temperatures where the rate of cell growth is reduced or prevented leading to eventual cell death. Cells are induced or genetically altered to overexpress cold-shock proteins and to enhance viability under low temperature conditions at which cell death normally occurs.
2. Description of Related Art
It is known that when certain cells are exposed to sudden changes in temperature that are either higher or lower than normal physiological temperatures, synthesis of specific proteins may be induced. When eukaryotic or prokaryotic cells are exposed to increases in temperature (termed "heat shock"), a shift occurs from the pattern of synthesis of normal cellular proteins to enhance the synthesis of a group of cell proteins commonly termed "heat-shock" proteins. Likewise, when certain cells (e.g. bacteria) are exposed to decreases in temperature, a class of proteins termed "cold-shock" proteins are synthesized at increased rates. It has been shown that a heat-shock response is induced to help protect the cell from otherwise lethal high temperatures. A number of these heat-shock proteins are molecular chaperones which can assist in refolding or degradation of heat-damaged cell proteins, and others are proteases or other components of the cell's proteolytic machinery which help the cell's ability to destroy the heat-damaged proteins.
Proteins have been identified as being produced at increased differential rates relative to other proteins upon the shifting of an E. coli. culture from ambient temperatures of 37.degree. C. to 10.degree. C. and lower, and have been termed "cold shock" proteins. See, Jones, P. G., VanBogelen, R. A., and Neidhardt, F. C. (1987) Journal of Bacteriology 169(5), 2092-2095; and Jones, P. G., Cashel, M., Glaser, G., and Neidhardt, F. C. (1992) Journal of Bacteriology 174(12), 3903-3914, each hereby incorporated by reference in their entirety. Some of the proteins are known to be involved in RNA transcription and translation, however, no specific in vivo beneficial effect of induction of the cold-shock proteins has been demonstrated.
Identification of proteins which prolong the life of the cell under conditions which the cell normally would not grow and would not remain viable is of major scientific and practical utility. Cells having this desirable characteristic would enable cell-based expression systems normally capable of operating only at room temperatures to operate at lower temperatures where certain proteins are produced more efficiently, in greater quantity or in greater activity. Such cell-based expression systems would advantageously be capable of expressing certain proteins which may undergo improper folding or degradation at higher temperatures, and so cannot be expressed at room temperatures.
Altering a cell to express proteins which enhance the cold-resistance characteristic of the cell is also advantageous in the field of agriculture to protect plants against frost or injury due to sudden low temperature; for production of food products, and any other biological (or pharmaceutical) products that should be protected from a harsh or stress-creating situation like extreme low temperatures. In addition, enhancing viability of mammalian cells at low temperatures may also be useful in cells or organ preservation, for example, where it is necessary to store the cell or organ prior to transplantation. Accordingly, the identification of proteins capable of enhancing or promoting cell viability at low temperatures where cell growth is greatly reduced or prevented and which lead to cell death and methods of altering cells to enhance their viability is of important scientific and commercial utility.
Trigger Factor ("TF") is an abundant protein in E. coli. whose in vivo importance has remained unclear for a long time. The amino acid sequence and corresponding DNA sequence has been determined and is shown in FIG. 1. See also Guthrie, B., and Wickner, W. (1990) Journal of Bacteriology 172(10), 5555-5562 hereby incorporated by reference in its entirety. It was originally isolated as a factor that bound to proOmpA protein and promoted its translocation into membrane vesicles in vitro. However, subsequent studies failed to demonstrate any role of TF in protein secretion in vivo. Recently, however, TF has been shown to have a number of remarkable properties including possible function as a molecular chaperone that promotes the folding of nascent polypeptides. TF is tightly associated with the 50S ribosomal particle and can be cross-linked to nascent polypeptide chains. In addition, TF was recently shown to be one of several E. coli. peptidyl-prolyl isomerases (PPI) that can catalyze the cis/trans isomerization of Xaa-Pro peptide bonds in polypeptides (PPI activity). This reaction is often a rate-limiting step in the folding of certain polypeptides, such as RNAseT1, especially at low temperatures.
TF has been shown to function together with the major chaperones, GroEL and GroES, in the selective degradation of certain polypeptides. See Kandror, O., Sherman, M., Rhode, M., and Goldberg, A. L. (1995) EMBO Journal 14(23), 6021-27. TF has also been shown to be a regulator of GroEL function. See Kandror, O., Sherman, M., Moerschall, R., and Goldberg, A. L. Journal of Biological Chemistry. A fraction of the cell's TF is associated with GroEL and these GroEL-TF complexes show much higher affinity for many unfolded proteins. Moreover, the addition of purified TF to GroEL in vitro increases GroEL's binding capacity for these proteins. This enhancement of GroEL binding can account for its ability to stimulate the degradation of certain proteins but may also be important in promoting protein folding.
Despite these seemingly important biochemical effects, increasing or decreasing TF levels was found not to affect growth rate or to have marked physiological consequences at 37.degree. C. The only clear in vivo effect was an increase in filamentation and mucoidity which was seen when TF levels were either increased or reduced. See Guthrie, B., and Wickner, W. (1990) Journal of Bacteriology 172(10), 5555-5562.
By contrast, the major molecular chaperones in E. coli. (e.g. Dna K and its cofactors, GroEL and GroES) are essential factors for normal growth at 37 .degree. C. They are also heat-shock proteins that are induced at high temperatures and by other conditions that cause damage to cell proteins. These chaperones prevent protein aggregation, help catalyze protein refolding, and can promote the selective degradation of heat-damaged polypeptides. Cells that fail to generate major heat-shock proteins are not able to grow at normal temperatures and die rapidly during heat-shock.
Unlike most molecular chaperones, TF is not a heat-shock protein and is not essential for viability at high temperatures. On the contrary, it has been demonstrated that the effects of TF on protein degradation (Kandror, O., Sherman, M., Rhode, M., and Goldberg, A. L. (1995) EMBO Journal 14(23), 6021-6027 hereby incorporated by reference in its entirety) and on GroEL's binding to proteins were much greater when cells were grown at 20.degree. C. than at 37.degree. C.
Despite attempts to elucidate the physiological importance of TF, the art provides no indication of whether TF is a cold-shock protein or what effect it may have in cells subjected to low temperatures where cell viability is decreased. Accordingly, a need exists to explore the nature of protein production induced by low temperatures at which cells normally would not grow and would not remain viable and to discover methods of increasing cell viability at such low temperatures.